Collagenase and Tyrosinase Inhibitory Effect of Isolated Constituents from the Moss Polytrichum formosum.

Mosses from the genus Polytrichum have been shown to contain rare benzonaphthoxanthenones compounds, and many of these have been reported to have important biological activities. In this study, extracts from Polytrichum formosum were analyzed in vitro for their inhibitory properties on collagenase and tyrosinase activity, two important cosmetic target enzymes involved respectively in skin aging and pigmentation. The 70% ethanol extract showed a dose-dependent inhibitory effect against collagenase (IC50 = 4.65 mg/mL). The methanol extract showed a mild inhibitory effect of 44% against tyrosinase at 5.33 mg/mL. Both extracts were investigated to find the constituents having a specific affinity to the enzyme targets collagenase and tyrosinase. The known compounds ohioensin A (1), ohioensin C (3), and communin B (4), together with nor-ohioensin D (2), a new benzonaphthoxanthenone, were isolated from P. formosum. Their structures were determined by mass spectrometry and NMR spectroscopy. Compounds 1 (IC50 = 71.99 µM) and 2 (IC50 = 167.33 µM) showed inhibitory activity against collagenase. Compound 1 also exhibited inhibition of 30% against tyrosinase activity at 200 µM. The binding mode of the active compounds was theoretically generated by an in-silico approach against the 3D structures of collagenase and tyrosinase. These current results present the potential application from the moss P. formosum as a new natural source of collagenase and tyrosinase inhibitors.

Author Correction: A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01488-x.

A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale.

Plant Milking Technology-An Innovative and Sustainable Process to Produce Highly Active Extracts from Plant Roots.

We have used an original technology (Plant Milking Technology) based on aeroponic cultivation of plants associated with the gentle recovery of active ingredients from roots. Extraction of bioactive molecules was achieved by soaking the roots, still attached to the living plants, into a nontoxic solvent for a 2 h period. This nondestructive recovery process allows using the same root biomass for successive harvesting dates, in a recyclable way. We have applied this technology to Morus alba L. (mulberry tree), an emblematic tree of the Traditional Chinese Medicine (TCM). Trees were aeroponically grown in large-scale devices (100 m2) and were submitted to nitrogen deprivation to increase the content in active molecules (prenylated flavonoids). The Plant Milking technology applied to Morus alba L. allowed to produce an extract enriched in prenylated compounds (18-fold increase when compared to commercial root extract). Prenylated flavonoids (moracenin A and B, kuwanon C, wittiorumin F, morusin) presented a high affinity for the aged-associated collagenase enzyme, which was confirmed by activity inhibition. In accordance, M. alba extract presents efficient properties to regulate the skin matrisome, which is critical during skin aging. The benefits have been especially confirmed in vivo on wrinkle reduction, in a clinical study that involved aged women. Plant Milking technology is an optimal solution to produce active ingredients from plant roots, including trees, that meet both customer expectations around sustainability, as well as the need for an efficient production system for biotechnologists.

The use of enzyme-coupled magnetic nanoparticles for studying the spectra of unusual substrates of mushroom tyrosinase by direct surface-assisted laser desorption/ionisation and high-resolution electrospray ionisation quadrupole-quadrupole-time-of-flight mass spectrometry.

RATIONALE: Tyrosinase-coupled magnetic particles (EMPs) were used to demonstrate that resorcinol-containing tyrosinase inhibitors are oxidised by tyrosinase only in the presence of the enzyme's classic substrate. This shows the potential for the application of EMPs as a non-organic matrix for monitoring enzymatic conversion of a novel substrate family directly on-the-spot, principally due to minimal enzyme requirement per analysis. METHODS: Tyrosinase was covalently coupled to core-shell-type silica-coated iron oxide magnetic nanoparticles (EMPs) that were applied as non-organic SALDI matrix suitable for studying low-mass compounds using a classic matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer. Because of the dual function of the EMPs - enzyme host and non-organic matrix - we describe this ionisation method as Enzyme-coupled Nanoparticles-Assisted LDI-MS (ENALDI-MS). Supplementary studies of the enzymatic conversion of glabridin and 3-(2,4-dihydroxyphenyl)propionic acid (DHPA) were conducted by high-resolution electrospray ionisation quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS). RESULTS: The initial experiment involving EMPs as non-organic matrix (ENALDI-MS) showed enzymatic conversion of glabridin, a strong tyrosinase inhibitor, only in the presence of L-Tyr, the classic tyrosinase substrate. These findings were evaluated by ESI-QqTOF-MS proving that glabridin and DHPA are converted into the corresponding quinones by tyrosinase only in the presence of the auxiliary monophenol or o-diphenol substrates (L-Tyr and catechin, respectively) capable of regenerating the active site of tyrosinase. CONCLUSIONS: EMPs were shown to be useful as a non-organic matrix to monitor enzymatic conversion of the novel tyrosinase substrate family directly on-the-spot with a minimal enzyme consumption (6.5 pmol/spot). Results obtained by ENALDI-MS were fully confirmed by ESI-QqTOF-MS demonstrating that resorcinol-containing tyrosinase inhibitors may be oxidised by the enzyme in the presence of its classic substrates.

Comparison of monolithic capillary electrochromatography and micellar electrokinetic chromatography for the separation of polycyclic aromatic hydrocarbons.

Atmospheric pollution of anthropic origin is recognized as a major risk factor for health, in particular for respiratory and cardio-vascular systems. Among these pollutants, polycyclic aromatic hydrocarbons (PAHs) are placed on the list of US Environmental Protection Agency (EPA) as 'priority' pollutants and four of them are assigned as potential carcinogens by The International Agency for Research on Cancer (IARC). In the present work two capillary techniques-micellar electrokinetic chromatography (MEKC) and monolithic capillary electrochromatography (CEC)-were compared for the separation of eleven PAHs. Both techniques compared in the present work are fully compatible with every standard apparatus of capillary electrophoresis. For MEKC, enhancement of selectivity and decrease of the separation window of eleven PAHs were obtained with methanol:borate 25 mM (20/80, v/v) running buffer containing 10 mM of hydroxypropylated γ-cyclodextrins with low SDS content (25 mM). In case of CEC, two acrylate-based monolithic stationary phases (MSPs) were evaluated for their application in the separation of eleven PAHs. The best MSP based on butyl acrylate was compared with MEKC in terms of sample capacity, PAHs elution order, LOQ, efficiency and effect of pH. Influence of the hydrophobicity of mobile phase on the PAHs elution order was also studied.

Enzyme-coupled nanoparticles-assisted laser desorption ionization mass spectrometry for searching for low-mass inhibitors of enzymes in complex mixtures.

In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: 'ion fading' (IF-ENALDI), based on superficial adsorption of inhibitors and 'ion hunting' (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme-inhibitor pairs: tyrosinase-glabridin and trypsin-leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin-leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

Splenic melanosis during normal murine C57BL/6 hair cycle and after chemotherapy.

Cancer chemotherapy is associated with serious side effects, including temporary hair loss and impairment of pigmentation. We suspect that ectopic melanin deposition occurring due to chemotherapy may add to these effects worsening the already unpleasant symptoms. We associated the ectopic occurrence of follicular melanin after chemotherapy with splenic melanosis - an interesting example of extradermal melanin localization - and we expected an increase in splenic melanin deposition after chemotherapy. Using the C57BL/6 murine model of synchronized hair cycle induced by depilation, we visualized splenic melanin by means of several histological and histochemical protocols of staining: hematoxylin and eosin, May-Grünwald-Giemsa and Fontana-Masson. Unexpectedly, the splenic deposition of melanin decreased due to application of cyclophosphamide (i.p. 120 mg/ kg body weight on day 9 post depilation). The drop was abrupt and lasted for at least 5 days (day 13-18 post depilation), as compared with normal hair cycle. Moreover, in mice with normal, depilation-induced hair cycle we observed a similar drop shortly before entering catagen (day 15 post depilation), followed by a slow and partial increase in splenic melanization up to day 27 post depilation in both groups. We conclude that cyclophosphamide negatively affects splenic melanization and/or extradermal transfer of ectopic melanin from the dystrophic hair follicles, but the most powerful down-regulator of splenic melanosis is normal and dystrophic catagen - the phase of hair follicle involution and re-modelling.

Substrate and inhibitor spectra of ethylbenzene dehydrogenase: perspectives on application potential and catalytic mechanism.

Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.

Continuous-flow step gradient mass spectrometry based method for the determination of kinetic parameters of immobilized mushroom tyrosinase in equilibrating conditions: comparison with free enzyme.

A mass spectrometry (MS)-based methodology for enzymatic assay in equilibrium conditions was designed and evaluated. This on-line assay involves the introduction of a continuous-flow step gradient (CFSG) of a substrate solution in the column containing immobilized enzyme and the simultaneous tracking of the product formation. We showed that the constant concentration of substrate in the entire bioreactor for an appropriate duration ensures the equilibration of the studied enzyme (mushroom tyrosinase). Under these conditions, it was demonstrated also that the kinetic and enzymatic parameters (Michaelis-Menten constant, K(M) , the maximal specific activity, SA(max)) are independent of the flow rate of the mobile phase. The feasibility of the mentioned approach for inhibitory tests was also investigated. The coupling of the mass spectrometer to the bio-reactor allows the selective monitoring of the enzymatic reaction products and increases their detection level. Very high sensitivity, 500 pmol/min/column, and selective monitoring of the products of the enzymatic reaction are allowed by MS detection. The methodology developed here constitutes a sensitive analytical tool to study enzymes requiring long equilibration times.

Extradermal melanin transfer? Lack of macroscopic spleen melanization in old C57BL/6 mice with de-synchronized hair cycle.

In quest of alternate, extradermal path of melanin transfer from skin to the visceral organs, we suggested that some portions of such melanin may be deposited in the spleen, which in young black C57BL/6 mice is often melanized. Here, we confirm these observation using young C57BL/6 female mice (up to 17 weeks) and show that this phenomenon cannot be observed in old animals where the hair cycle is not synchronized any more. The experiments were carried out both on spontaneous and depilation-induced hair cycle. We have checked it as a side-observation over many other experiments carried out on young and old C57BL/6 female mice (up to 2.5 years of life). The presence or absence of melanin in the spleens was checked macroscopically, and histologically by Fontana-Masson (FM) staining, and synchronization of the hair cycle - by standard histomorphometric analysis of the back skin hair follicles. In about 40% of old spleens black FM-stainable "debris" could be found under closer histological examination. This study shows that, at least in part, the phenomenon of splenic melanosis in C57BL/6 mice can be correlated with the synchronized skin melanization parallel to the hair cycle progress, and that splenic melanin undergoes gradual degradation during the mouse life.